Biosynthetic human growth hormone

ABSTRACT

A desired protein having the formula: 
     
         A-B-C-P 
    
     wherein 
     a) A is Lys or Arg, and B and C are arbitrary amino acids, or 
     b) A is an arbitrary amino acid different from Pro, Lys and Arg, and B and/or C is Pro, 
     is produced from a biosynthetically formed amino acid extended protein having the formula: 
     
         X-A-B-C-P 
    
     wherein A, B, C and P are as defined above, and X is an amino acid sequence with an even number of amino acids, of which the first one, seen from the N-terminal end, is different from Lys and Arg, all other uneven amino acids are different from Pro, Lys and Arg, and all even amino acids are different from Pro, by reaction with the enzyme dipeptidyl aminopeptidase (DAP I). The desired protein is obtained in a pure state. Thus, e.g. hGH without content of Met-hGH may be produced by the process.

This is a continuation of application Ser. No. 372,692, filed Jan. 13,1995, which is, in turn, a continuation of application Ser. No. 959,856,filed Nov. 12, 1992, now abandoned, which is, in turn, a continuation ofapplication Ser. No. 759,106, filed Sep. 6, 1991, now abandoned, whichis, in turn, a continuation of application Ser. No. 215,602, filed Jul.1, 1988, now abandoned, which is, in turn, a continuation-in-part ofapplication Ser. No. 910,230, filed as PCT/DK86/00014, Feb. 6, 1986, nowabandoned, which is, in turn, a continuation-in-part of application Ser.No. 640,081 filed Aug. 8, 1984, now abandoned, which is in turn acontinuation-in-part of International Application PCT/DK83/00118, filedDec. 9, 1983.

The present invention concerns a process for preparing a desired proteinhaving the formula stated in the introductory portion of claim 1.

It is known from the U.S. Patent Specification 4 342 832 to producebiosynthetic hGH by fermentation of a recombinant host cell, inparticular E. Coli, which codes for hGH with associated methionine.However, this known process results in hGH whose N terminus has attachedto it the amino acid methionine which is not present in ripe hGH.

Owing to the risk of antigenic reactions and other side effects in theuse of a growth hormone which is not quite identical with hGH, it isinexpedient to use biosynthetic Met-hGH.

Accordingly, there is a great need for a process which enablesproduction of biosynthetic hGH with a correct amino acid sequence. Asolution to this problem has been proposed by U.S. Ser. No. 488 232 (DKPatent Application 2046/84), which concerns a process for producing hGHfrom pre-hGH in a recombinant prokaryotic microorganism, such asPseudomonas aeruginosa or E. coli.

The use of Ps. aeruginosa for the production of hGH without methioninefor therapeutic use, however, is vitiated by the risk that thisbacterium and many other Pseodomonas bacteria, which are potentiallypathogenic, synthesize toxic toxins which are problematic.

The expression of pre-hGH followed by proteolytic cleavage to obtainripe hGH in E. coli (which is not pathogenic) is indicated in DK PatentApplication 2046/84, but it is not documented in that specification thatthe proteolytic cleavage unambiguously leads to the formation of ripehGH, i.e. with a correct amino acid sequence.

As mentioned above; risks may be involved in using Met-hGH. Thoughmethods have been proposed for enzymatic cleavage of the methioninegroup by means of aminopeptidases, the problem would not be solved bythis because the known enzymatic processes of this type do not lead to a100% conversion. A mixture of hGH and Met-hGH would occur, which cannotbe separated completely by conventional preparative purificationprocesses.

The present invention is based on the finding that the enzyme dipeptidylaminopeptidase I (DAP I) or cathepsin C (EC(3,4,14,1)) is suitable forcleaving an N-terminal amino acid sequence with an even number of aminoacids to form a desired protein having the formula:

    A-B-C-P

wherein

a) A is Lys or Arg, and B and C are arbitrary amino acids, or

b) A is an arbitrary amino acid different from Pro, Lys and Arg, and Band/or C is Pro,

and P is in both cases the residual amino acid sequence in the desiredprotein.

Thus, DAP I has been found suitable not only for production of hGH inwhich the three first amino acids are Phe-Pro-Thr, but proteins ingeneral which satisfy the conditions of the sequence A-B-C-P.

Thus, the process of the invention is characterized in that abiosynthetically formed amino terminal extended protein having theformula:

    X-A-B-C-P,

wherein A, B, C and P are as defined above, and X is an amino acidsequence having an even number of amino acids, of which the first one,seen from the N-terminal end, is different from Lys and Arg, all otheruneven amino acids are different from Pro, Lys and Arg, and all evenamino acids are different from Pro, is reacted with the enzymedipeptidyl aminopeptidase I (DAP I).

Examples of proteins which may be produced by the process of theinvention are the following:

Proteins with lysine in the first site

    ______________________________________                                        Name          Origin       N-terminal sequence                                ______________________________________                                        Cholecystokinin                                                                             Porcine      Lys--Ala--Pro--                                    Neurotoxin I  Scorpion     Lys--Asp--Gly--                                    Penicillinase Staphylococcus                                                                             Lys--Glu--Leu--                                                  Aureus                                                          Ribonuclease  Bovine       Lys--Glu--Ser--                                    Proparathyrin Human        Lys--Ser--Val--                                    Lactalbumin   Human        Lys--Glu--Phe--                                    Kallidin II   Human        Lys--Arg--Pro--                                    Purothionine A-I                                                                            Wheat        Lys--Ser--Cys--                                    Viscotoxin A3 Eru. Mistelten                                                                             Lys--Ser--Cys--                                    Lysozyme      Human        Lys--Val--Phe                                      ______________________________________                                    

Proteins with arginine in the first site

    ______________________________________                                        Name          Origin       N-terminal sequence                                ______________________________________                                        Beta Casein   Bovine       Arg--Glu--Leu--                                    Posterior Pituitary                                                                         Bovine       Arg--Gly--Glu--                                    Peptide                                                                       Serum Albumin Bovine       Arg--Gly--Val--                                    Precursor                                                                     Long Neurotoxin I                                                                           Black Mamba  Arg--Thr--Cys--                                    Tuberculin-Active                                                                           Mycobacterium                                                                              Arg--Leu--Leu                                      Protein       Tuberculosis                                                    Bradykinin (Kalli-                                                                          Bovine       Arg--Pro--Pro                                      din I)                                                                        Amyloid Protein AA                                                                          Human        Arg--Ser--Phe--                                    ______________________________________                                    

Proteins with proline in the second site

    ______________________________________                                        Name            Origin    N-terminal sequence                                 ______________________________________                                        Choriogonadotropin                                                                            Human     Ala--Pro--Asx--                                     Follitropin (α-chain)                                                                   Human     Ala--Pro--Asp--                                     Pancreatic Hormone                                                                            Bovine    Ala--Pro--Lys--                                     Aspartate Aminotrans-                                                                         Porcine   Ala--Pro--Pro--                                     ferase                                                                        Plasminogen     Human     Glu--Pro--Leu--                                     Insulin-like    Human     Gly--Pro--Glu--                                     Growth Hormone                                                                Prealbumin      Human     Gly--Pro--Thr--                                     Prolactin       Porcine   Leu--Pro--Ile--                                     Lipid-binding Pro-                                                                            Human     Thr--Pro--Asp--                                     tein C-I                                                                      Cholera Enterotoxin                                                                           Vibrio    Thr--Pro--Glu--                                     (β-chain)  Cholerae                                                      Prolactin       Bovine    Thr--Pro--Val--                                     Lymphotoxin     Human     Lys--Pro--Gly--                                     Interleukin-2   Human     Ala--Pro--Thr--                                     Erythropoietin  Human     Ala--Pro--Pro--                                     ______________________________________                                    

Proteins with proline in the third site

    ______________________________________                                        Name          Origin       N-terminal sequence                                ______________________________________                                        Neurocarzinostatin                                                                          Streptomyces Ala--Ala--Pro--                                                  Carzinostaticus                                                 Somatotropin  Bovine       Ala--Phe--Pro--                                    Carbonic Anhydrase B                                                                        Human        Ala--Ser--Pro--                                    Toxin II      Sea Anemone  Gly--Val--Pro--                                    Allergin RA5  Wormwood     Leu--Val--Pro--                                    Lac Repressor E. coli      Met--Lys--Pro--                                    Alcohol Dehydrogenase                                                                       Yeast        Ser--Ile--Pro--                                    Orosomukoid   Human        Glx--Ile--Pro--                                    Interleukin-1 Murin        Ser--Ala--Pro--                                    ______________________________________                                    

Examples of starting materials which may be cleaved with DAP I are thefollowing:

    ______________________________________                                        Met--Glu--Ala--Glu                                                                          hGH                                                             Met--Phe--Glu--Glu                                                                          hGH       to obtain hGH                                         Met--Thr--Glu--Glu                                                                          hGH       (proline in the second site)                          Met--Glu--Glu--Glu                                                                          hGH                                                             Ala--Ala--Glu--Glu                                                                          hGH                                                             Met--Phe--    Glu-hGH                                                         Met--Leu--    Glu-hGH   to obtain Glu-hGH                                     Ala--Glu      Glu-hGH   (proline in the third site)                           Met--Ala--    Glu-hGH                                                         ______________________________________                                    

The present process is thus suitable for production of biosyntheticproteins, such as hGH having attached to it a pre-sequence which can becleaved enzymatically in a high yield, and which gives products by theenzymatic cleavage which may be separated satisfactorily by knownpurification methods, such as ion exchange.

The biosynthetic ripe hGH produced is free of all non-hGH pituitaryrelated contaminants including those which cause Creutzfeldt-Jakobdisease, and may cause Gerstmann-Str aussler-Scheinker Syndrome andKuru, by virtue of its biosynthetic production.

Examples of suitable amino terminal extensions which may be cleaved bymeans of DAP I are those in which the last amino acid in the amino acidsequence X, before A, is an amino acid with a charged side chain, suchas Glu or Asp.

These amino terminal extensions may be obtained by fermentation in asuitable substrate of a microorganism which is transformed with aplasmid ceding for the desired extended protein.

After expression, the methionine residue is optionally cleavedenzymatically in the microorganism so that the recovered protein isattached to the desired amino terminal extension with an even number ofamino acids which may be cleaved selectively and in a high yield.Isolation of the resulting protein takes place in a manner known per se,e.g. by chromatographic methods.

By selecting an amino extension which contains at least one amino acidwith a charged side chain, such as a carboxyl group, it is possible toperform the separation and the purification of amino terminal extendedprotein from the ripe protein.

At least one of the charged amino acids may be attached directly to theN-terminal end of the protein because it may then be observed whetherthe entire amino terminal extension has been cleaved. This isparticularly important when the microorganism in vivo only partlycleaves the N-terminal methionine residue.

It is most expedient that an amino acid with charged side chains in theamino terminal extension to the protein is either exclusively positivelyor negatively charged. This prevents amino terminal extended protein,partly enzymatically converted amino terminal extended protein andauthentic protein from having the same net charge at any time.

In hGH, slight deamidation of certain Gln and Asn residues takes place,i.e. Gln and Asn are converted to Glu and Asp, respectively--i.e. aminoacids with negatively charged side chains. For this reason it willtherefore be most expedient that the charged amino acid in the aminoterminal extension are the negatively charged Glu and/or Asp, becausethis avoids the situation of one or more deamidations in hGHneutralizing the positive charge/charges present in the extension. Suchneutralization of charges will make it impossible to separate possiblyunreacted deamided amino terminal extended hGH by ion exchange from theenzymatically formed hGH.

Examples of particularly suitable amino terminal exten- sions which maybe cleaved with DAP I are

1. Met-Glu-Ala-Glu

2. (Ala-Glu)_(r), wherein r is an integer from 1 to 12

3. Met-Phe-Glu-Glu

4. Thr-Glu-Ala-Glu

5. Met-Asp-Ala-Asp

6. Met-Glu-Ala-Asp

These and other suitable amino terminal extensions may be obtained byfermenting in a suitable substrate a microorganism transformed with aplasmid, which codes for the desired protein with these attached aminoterminal extensions.

In some specific pre-sequences, methionine, which is the N-terminalamino acid in all proteins formed in E. coli, is cleaved enzymaticallyin the microorganism after expression of the protein. This results e.g.in the above-mentioned amino terminal extended proteins.

These proteins are purified by conventional purification methods. Theamino terminal extension is cleaved selectively and in a high yield. Theformed protein may then easily be separated from any residues of partlyconverted amino terminal extended protein by known chromatographicmethods.

The process of the invention will be illustrated more fully below bymeans of some working examples.

EXAMPLE 1 Preparation of hGH by means of DAP I

A cloned DNA sequence which codes for a protein having an amino acidsequence like human growth hormone, hGH (191 amino acid residues, thefirst four amino acids of which are Phe-Pro-Thr-Ile) is coupled with thefollowing synthetically produced, dual-stranded DNA sequence so that the3' end of the + strand is coupled to the +5' end of the above-mentionedgene, and the 5' end of the synthetic DNA sequence strand is coupled tothe 3' end of the above-mentioned gene by blunt end ligature

    +5' CGATG GCT GAA

    -3' TAC CGA CTT

where the 2 first nucleotides in the + strand are a ClaI restrictionsite overhang, and the following nucleotide sequences code for the aminoacids Met-Ala-Glu-.

The above-mentioned gene is introduced by ordinary gene cloningtechniques into an expression plasmid containing a fused Trp-Lacpromotor as well as the SD sequence AGGA. This structure expressesMet-Ala-Glu-hGH.

This plasmid structure is then introduced into an E. coli cell by priorart techniques. A suitable clone containing the above-mentionedstructure is isolated and cultivated in a 5:1 scale. The cells wereharvested by centrifugation and are suspended in a small volume andlyzated using a so-called "French press".

The expected fusion protein could be demonstrated in the above-mentionedbacterial extract by immunological methods using hGH antibodies,corresponding to a concentration of 200 mg/l in the culture medium.

The fusion protein is purified conventionally by anion exhange, ammoniumsulfate precipitation and hydrophobic chromatography.

The purified Met-Ala-Glu-hGH was evaluated to be more than 99% pure,evaluated by SDS electrophoresis.

An amino terminal sequence determination showed that the purified hGhmaterial had the sequence Ala-Glu-hGH, which means that Met has beencleaved by an E. coli enzyme.

100 mg of AE-hGH in 10 mM Tris-Cl. pH 4.2 (1.5 mg/ml) were admixed with5 mg of DAP I (3,4,14,1).

The reaction mixture was then incubated at 40° C. After 4 1/2 hours themixture was cooled to 4° C. The cooled reaction mixture was thenfractionated by anion exchange, and following this the main peak (hGHproduct) was isolated. The yield was 90%.

The hGH product was shown to be more than 99% pure, evaluated by SDSelectrophoresis. An amino terminal determination (Edman degradation)showed that the amino terminal sequence of the hGH product wasPhe-Pro-Thr-Ile-Pro-, i.e. as for authentic hGH.

The biological activity of the hGH product was determined by a tibiatest and was found to be 2.5 IU/mg, which is also the case withauthentic hGH.

EXAMPLE 2 Preparation of hGH from Met-Glu-Ala-Glu-hGH with DipeptidylAminopeptidase I, (DAP I)

Met-GIu-Ala-GIu-hGh is produced by gene techniques in principle asdescribed in example 1. Met-Glu-Ala-Glu-hGH is purified from thefermentation product by anion exchange and hydrophobic interactionchromatography.

The purified Met-Glu-Ala-Glu-hGh was evaluated to be more than 99% pureby ion exchange and SDS electrophoresis.

An amino terminal sequence determination showed that the purified hGHhad the sequence Met-Glu-Ala-Glu-Phe-Pro-Thr-Ile-Pro-Leu, where the lastsix amino acids correspond to the N-terminus in hGH. 200 ml ofMet-Glu-Ala-Glu-hGH (2.0 mg/ml) in 20 mM Tris, 10 mM citric acid, 25 mMNacl, pH 5.2 were admixed with 10,000 mU (corresponding to 3.3 mg)dipeptidyl aminopeptidase I (E.C. 3,4,14,1) from Boehringer Mannheim.Other makes may be used as well. The pH value is optionally readjustedto 4.2.

The reaction mixture was then incubated at 40° C. for 60 minutes,resulting in a more than 98% conversion of Met-Glu-Ala-Glu-hGh to hGH.The reaction mixture was cooled to 4° C. after completed reaction. Thefurther purification comprises isoprecipitation, gel filtration and ananion exchange.

The hGH product was shown to be more than 99% pure evaluated by IE-HPLCand SDS electrophoresis. An amino terminal sequence determination byEdman degradation showed that the amino terminal sequence of the HGHproduct was Phe-Pro-Thr-Ile-Pro-Leu, i.e. as for authentic hGH.

The biological activity of the hGH product was determined by a tibiatest and was found to be equipotent with pituitary hGH.

EXAMPLE 3 Preparation of hGH from Met-Phe-Glu-Glu-hGH with DipeptidylAminopeptidase I

Met-Phe-Glu-Glu-hGh is produced by gene techniques in principle asdescribed in example 1. Met-Phe-Glu-Glu-hGH is purified from thefermentation product by anion exchange and hydrophobic interactionchromatography.

The purified Met-Phe-Glu-Glu-hGH was evaluated to be more than 99% pureby IE-HPLC and SDS electrophoresis.

An amino terminal sequence determination showed that the purified hGHproduct had the sequence Met-Phe-GIu-Glu-Phr-Thr-Ile-Pro-Leu, where thelast six amino acids correspond to the N-terminus in hGH.

100 ml of Met-Phe-Glu-Glu-hGH (1.5 mg/ml) in 20 mM Tris, 10 mM citricacid, 25 mM NaCl, 1 mM L-Cysteine pH 4.2 were admixed with 15,000 mU(corresponding to 5.0 mg) aminopeptidase I (E.C. 3,4,14,1) fromBoehringer Mannheim. Other makes may be used as well. The pH value isoptionally readjusted to 4.2.

The reaction mixture was then incubated at 40° C. for 60 minutes,resulting in a more than 98% conversion of Met-Phe-Glu-Glu-hGH to hGH.The reaction mixture was cooled to 4° C. after completed reaction. Thefurther purification comprises isoprecipitation, gel filtration and ananion exchange.

The hGH product was shown to be more than 99% pure evaluated by IE-HPLCand SDS electrophoresis. An amino terminal sequence determination byEdman degradation showed that the amino terminal sequence of the hGHproduct was Phe-Pro-Thr-Ile-Pro-Leu, i.e. as for authentic hGH.

The biological activity of the hGH product was determined by a tibiatest and was found to be equipotent with pituitary hGH.

EXAMPLE 4 Preparation of hGH from Ala-Glu-Ala-Glu-hGH with DipeptidylAminopeptidase I

Met-Ala-Glu-Ala-Glu-hGH is produced by gene techniques in principle asdescribed in example 1. Met is cleaved in vivo so that the proteinformed by fermentation is Ala-Glu-Ala-Glu-hGH. This is purifiedconventionally by anion exchange and hydrophobic interactionchromatography.

The purified Ala-Glu-Ala-Glu-hGH was evaluated to be more than 99% pureby IE-HPLC and SDS electrophoresis.

An amino terminal sequence determination showed that the purified hGHproduct had the sequence Ala-Glu-Ala-Glu-Phe-Pro-Thr-Ile-Leu-Pro-Leu,where the last six amino acids correspond to the N-terminus in hGH.

100 ml of Ala-Glu-Ala-Glu-hGH (2.0 mg/ml) in 20 mM Tris, 10 mM citricacid, 25 mM NaCl, pH 4.2 were admixed with 20,000 mU (corresponding to6.7 mg) Dipeptidyl Aminopeptidase I (E.C. 3,4,14,1) from BoehringerMannheim. Other makes may be used as well. The pH value is optionallyreadjusted to 4.2.

The reaction mixture was then incubated at 40° C. for 60 minutes,resulting in a more than 98% conversion of Ala-Glu-Ala-Glu-hGH to hGH.The reaction mixture was cooled to 4° C. after completed reaction. Thefurther purification comprises isoprecipitation, gel filtration and ananion exchange.

The hGH product was shown to be more than 99% pure evaluated by IE-HPLCand SDS electrophoresis. An amino terminal sequence determination byEdman degradation showed that the amino terminal sequence of the hGHproduct was Phe-Pro-Thr-Ile-Pro-Leu, i.e. as for authentic hGH.

EXAMPLE 5 Preparation of ILIβ from Met-Glu-Ala-Glu-ILIβ

Biosynthetically produced Met-Glu-Ala-Glu-ILIβ was purified and isolatedby chromatography, and the eluate was admixed with 0.38 unit of DAP I(from Boehringer Mannheim, called cathepsin C, 21.9 IU/ml) per mg ofprotein, calculated on the basis of E (280, 0.1%)=0.6. The reactionmixture was left to stand for 45 min. at 37° C. The solution wasdialyzed against 20 mM Na-citrate, 2 mM EDTA, pH=4.0 at 4° C. for 18hours.

The dialysate was applied to an FF-Q Sepharose CL6B column in Tris-Cl pH=8.0 with an NaCl gradient to 0.2M.

The ILIβ fraction was concentrated by ultrafiltration with a 10 ml Novacell to a volume of 2.0 ml (c=7.0 mg per ml). The pooled concentrate wasapplied to a Sephacryl column in 0.5M Na-acetate, pH=3.5.

The product was characterized by amino acid analysis and N-terminalsequence analysis. The sequence was shown to be identical with the first42 N-terminal amino acids in authentic ILIβ.

EXAMPLE 6 Preparation of human lysozyme (hLZ)

Usual biotechnological methods are used for preparation of the gene forthe protein MFEE-hLZ, where hLZ has the amino acid sequence:

1 K V F E R C E L A R T L K R L G M D G Y R G I S L A N W M C

31 L A K W E S G Y N T R A T N Y N A G D R S T D Y G I F Q I N

61 S R Y W C N D G K T P G A V N A C H L S C S A L L Q D N I A

91 D A V A C A K R V V R D P Q G I R A W V A W R N R C Q N R D

121 V R Q Y V Q G C G V *

The gene is introduced into a suitable expression system and cultivatedto form MFEE-hLZ. This protein was purified and treated with DAP I underthe conditions stated in example 1. Thereby, authentic pure humanlysozyme is isolated.

EXAMPLE 7 Preparation of IGF-1

Usual biotechnological methods are used for the preparation of a plasmidwhich codes for an extended human insulin-like growth factor 1 havingthe formula Met-Phe-Glu-Glu-IGF-1, where the sequence IGF has thefollowing structure: ##STR1##

The plasmid is introduced into E. coli, which is cultivated under usualconditions. The formed fusion product is isolated and purified in aknown manner and treated with the enzyme DAP I to form authentic humanIGF-1.

EXAMPLE 8 Preparation of bovine growth factor, bGH

Usual biotechnological methods are used for the preparation of plasmidwhich codes for an extended bovine growth hormone having the formulaMFEE-bGH, where the sequence bGH has the following structure:

1 A F P A M S L S G L F A N A V L R A Q H L H Q L A A D T F K

31 E F E R T Y I P E G Q R Y S I Q N T Q V A F C F S E T I P A

61 P T G K N E A Q Q K S D L E L L R I S L L L I Q S W L G P L

91 Q F L S R V F T N S L V F G T S D R V Y E K L K D L E E G I

121 L A L M R E L E D G T P R A G Q I L K Q T Y D K F D T N M R

151 S D D A L L K N Y G L L S C F R K D L H K T E T Y L R V M K

181 C R R F G E A S C A F *

The plasmid is introduced into E. coli, which is cultivated under usualconditions. The formed fusion product is isolated and purified bychromatographic methods, followed by a treatment with the enzyme DAP I.The reaction mixture was processed to develop pure bGH.

EXAMPLE 9 Preparation of pickwale ribonuclease, pwR

Usual biotechnological methods are used for the preparation of a plasmidwhich codes for an extended protein having the formula MFEE-pwR, wherethe sequence pwR has the following structure:

1 R E S P A M K T Q R Q H M D S G N S P G N N P N Y C N Q M M

31 M R R K M T Q G R C K P V N T F V H E S L E D V K A V C S Q

61 K N V L C K N G R T N C Y E S N S T M H I T D C R Q T G S S

91 K Y P N C A Y K T S Q K E K H I I V A C E G N P Y V P V H F

121 D N S V *

The plasmid is introduced into E. coli, which is cultivated under usualconditions. The formed fusion product is isolated and purifiedchromatographically, and it is treated with the enzyme DAP I. Thereaction mixture is processed to isolate pure pwR.

I claim:
 1. Biosynthetic ripe human growth hormone free of contaminantsfrom pituitary derived human growth hormone.
 2. Biosynthetic ripe humangrowth hormone produced by expressing an amino terminal extended humangrowth hormone fusion protein in a microorganism capable of suchexpression, enzymatically cleaving the amino terminal extension andrecovering the biosynthetically produced ripe human growth hormone.